Fig 1.
Recordings of the waveforms of the atrioventricular inflows in masu and cherry salmon using pulsed-wave echocardiography.
(A, B) Representative Oncorhynchus masou masou pictures: (A) masu salmon at 29 months post fertilization, (B) cherry salmon reared in seawater for five months after being reared in freshwater for 24 months. Scale bars = 10 cm. (C, D, E) Representative velocity waveforms of atrioventricular inflows in the masu (C) and cherry salmon of monophasic (D) and biphasic patterns (E). Blood passing the atrioventricular valve was observed using pulsed-wave Doppler echocardiography simultaneously with electrocardiography. Green arrowheads indicate P waves. Black left–right double arrows indicate R-R intervals. a: atrial systolic velocity, e/a: fusion velocity of atrial systolic and early diastolic inflows, e: early diastolic velocity. (F) Peak wave velocity. ○: masu salmon atrial systolic velocity (31.3 ± 6.4 cm/s, N = 37 peaks from five fish), ▲: cherry salmon early diastolic waves and atrial systolic waves (e/a) fusion velocity (36.7 ± 3.6 cm/s, N = 12 peaks from two fish), ■: cherry salmon early diastolic velocity (16.5 ± 2.6 cm/s, N = 15 peaks from two fish), ●: cherry salmon atrial systolic velocity (34.7 ± 4.2 cm/s, N = 15 peaks from two fish). Lines and error bars indicate the mean ± standard deviation. Minimal data sets for atrioventricular velocities are found in S1 File.
Table 1.
Body size and heart mass measurements (means ± standard deviation) for Oncorhynchus masou masou.
Fig 2.
Relative ventricular stiffness of masu and cherry salmon.
(A) The normalized end-diastolic pressure–volume relationship (EDPVR) at 30 mpf for masu and cherry salmon. The horizontal axis shows the ventricular volume normalized by the mass of the masu (N = 4) and cherry salmon ventricles (N = 3). The vertical axis shows the ventricular pressure. Ventricular mass is shown in S5B Fig. (B) Scores for ventricular stiffness in the masu and cherry salmon. The results of the EDPVR (A) were appended in Eq (1) to obtain the exponential C. Black horizontal lines in (B) indicate the means. ○: masu salmon, ●: cherry salmon.
Fig 3.
Histological analysis of masu and cherry salmon hearts.
(A, B) Representative images of masu (A) and cherry (B) salmon hearts 29 mpf. At: atrium, V: ventricle, Ba: bulbus arteriosus. (C–J) Elastica van Gieson staining images of the sagittal sections of the hearts of masu and cherry salmon 29 mpf. (C, G) Images of the whole hearts. (D, H) Magnified images of the ventricles. Black double arrows indicate the thickness of the compact layer. Black arrowheads indicate coronary vessels. (E, I) Higher magnification images of the compact layers. (F, J) Higher magnification images of the spongy layers. Collagen fibers: bright red; cytoplasm: yellow; elastic fibers: purple-black; nuclei: dark black. Cl: compact layer, Sl: spongy layer. Scale bars = 1 cm in (A) and (B); 5 mm in (C) and (G); 1 mm in (D) and (H); 100 μm in (E), (I), (F), and (J). (K) Percentage compact layer in the sagittal ventricle sections. (L) The number of nuclei per yellow area in the compact layer. (M) The collagen fiber area percentage in the compact layer. Bar graphs and error bars indicate the means and standard deviations, respectively. ○: masu salmon, ●: cherry salmon. Minimal data sets of (K–M) are found in S3 File.
Fig 4.
Morphology and sizes of the isolated cardiomyocytes.
(A, B) Representative bright-field images of the primary cultured cardiomyocytes of masu and cherry salmon 29 mpf. Scale bars = 20 μm. (C) Length of the long axis of the isolated cardiomyocytes; masu salmon (56 ± 19 μm, N = 55), cherry salmon (58 ± 20 μm, N = 50). (D) Area of the isolated cardiomyocytes; masu salmon (559 ± 239 μm2, N = 55), cherry salmon (518 ± 197 μm2, N = 50). Lines and error bars indicate the mean ± standard deviation. ○: masu salmon, ●: cherry salmon. Actual measurements of cardiomyocyte morphology are shown in the S4 File.
Fig 5.
Molecular weights of the Connectin isoforms and sarcomere lengths in the hearts of masu and cherry salmon.
(A) A representative CBB-stained gel image. Lanes 2–7 show the molecular weights of Connectin isoforms in the atrium, ventricle, and bulbus arteriosus of masu salmon (Ma) and cherry salmon (Ch). The following samples were used as molecular weight standards: a major Connectin isoform in zebrafish heart (Zf, 3500 kDa) in lane 8; Connectin N2A isoform from the skeletal muscles of mice (M1, 3700 kDa) in lanes 1 and 10; the Connectin N2B isoform expressed in the left ventricle of mice (M2, 3000 kDa) in lane 9. α-connectin (T1) and β-connectin (T2) indicate an intact Connectin and its degraded product, respectively. MHC indicates Myosin heavy chain (molecular weight ~220 kDa each), which was used as a loading control. (B) Electropherogram of lanes 2–7 of the CBB-stained gel in (A). Black arrowheads indicate the band peaks for the ventricles of masu and cherry salmon in the α-connectin zone. (C–F) Representative transmission electron microscopy images of the cross-sections and longitudinal sections in the ventricular sarcomeres of masu and cherry salmon 29 months post fertilization. Insets of (C, E) showing higher magnification images of the cross-sections of myofibril bundles. Scale bars = 500 nm in (C) and (E) and 1 μm in (D) and (F). (G) The ventricular sarcomere lengths of the z-line to z-line in the ventricles of masu salmon (2.06 ± 0.08 μm, N = 42) and cherry salmon (2.05 ± 0.11 μm, N = 90). A black double arrow indicates sarcomere length. Lines and error bars indicate the means ± standard deviations. ○: masu salmon, ●: cherry salmon. A raw data image of CBB-stained gel is found in S1 Raw images. Minimal data sets for (G) are found in S5 File.